A goal of the Medical Virology Section since the early 1980s has been the elucidation of the mechanisms that permit and regulate the latency and reactivation of the neurotropic human alphaherpesviruses, herpes simplex viruses (HSV) 1 and 2 and the varicella-zoster virus (VZV). These efforts were informed by the rich patient experience being acquired by Section staff through their parallel clinical studies of disease natural history, asymptomatic virus shedding and transmission, and responsiveness to experimental antiviral therapies and candidate vaccines. The dominant thrust of work over the past several years has been to define the cellular distribution of latent VZV DNA in human ganglia and to delineate the biological roles of HSV latency-associated transcripts (LATs). Most work by others has involved the molecular characterization of HSV-1 LATs and their study through creation of mutants bearing targeted deletions in their promoter or transcription domains. This laboratory adopted a different strategy: we study LAT expression and biology in HSV-2 because it is a more important human pathogen, and because the guinea pig model of genital infection is the only one that evinces spontaneous disease reactivation. By 1998 we had defined the promoter elements for the HSV2 LATs, deleted all or some of them from the virus, and studied these mutants using ocular and genital models of infection in mice and guinea pigs wherein it is possible to follow the clinical and virological course of acute infection and spread to regional ganglia, to quantify copies of viral genomes in acutely and latently infected tissues, and to induce reactivation upon ex vivo co-cultivation or by in vivo exposure of animals to heat or ultraviolet light. The work revealed that HSV2 deleted fully for expression of LATs infected animals and established latency normally, but it is impaired for reactivation. Moreover, the Section staff showed that the quantity of latent viral DNA is an essential arbiter of rates of subsequent spontaneous reactivation. Efforts began then to refine techniques for localizing and quantifying latent viral sequences. Scientists in the Section were the first in the field to introduce and standardize methods for real-time PCR using the Taqman system, for isolation of individual cells using laser capture microdissection (LCM), and for creation, breeding and study of mice transgenic for latency sequences. Analyses of these transgenic mice showed that expression of the HSV2 latency region as a transgene had no impact on the course of acute exogenous infection with HSV2, on the establishment of latency or on the potential for reactivation. Work over the past year involved refinement and utilization of a combination of in situ hybridization, LCM, and real-time PCR techniques to determine the cellular distribution of latent HSV-1 and VZV DNA and HSV-1 latency-associated transcripts (LAT) in human sensory ganglia at the single cell level. We found that many more neurons in human ganglia harbor HSV-1 DNA (2-11%) than are positive by in situ hybridization for LATs (0.2-1.5%). The mean number of copies of HSV-1 DNA per cell was 11.3. In fact, most of the HSV-1 DNA positive neurons are LAT-negative by in situ hybridization. We found that HSV-1 DNA was virutally exclusive to neurons and was rarely present in the adjacent satellite cells. The viral DNA load in a neuron is an important, but not the only, determinant of the accumulation of LAT in that neuron. We also found that latent VZV DNA is located in 1.0-6.9% of neurons but rarely, if ever, in satellite cells, contrary to our findings of 16 years ago using in situ hybridization. THe mean number of copies of VZV DNA per cell was 6.9.